The present invention relates to a novel polyhydroxyalkanoate (may be abbreviated as PHA, hereinafter). Also, it relates to a very effective method for manufacturing such PHA using microorganisms capable of producing PHA and accumulating it in the cell.
Hitherto, many microorganisms have reportedly produced poly-3-hydroxybutyric acid (may be abbreviated as PHB) or other PHAs and accumulated them in the cell (xe2x80x9cBiodegradation Plastic Handbookxe2x80x9d, Biodegradable Plastic Research Association, NTS, Co. Ltd., P178-197). As in the case of conventional plastics, these polymers can be used for producing various kinds of products through melt processing and the like. Furthermore, since they are biodegradable they are advantageously degraded completely by microorganisms in the natural world, and do not remain in the natural environment to cause pollution unlike many conventional synthetic polymers. Also, they have good biocompatibility, and applications as medical soft materials and the like are expected.
It is known that these PHAs may have various compositions and structures depending on types of microorganisms for use in production thereof and culture medium compositions, culture conditions and the like, and until now, studies have been made on control of these compositions and structures, principally in terms of improvement of properties of PHA.
For example, it has been reported that Alcaligenes eutropus H16, ATCC No. 17699 and its mutants produce copolymers of 3-hydroxybutyric acid (may be abbreviated as 3HB, hereinafter) and 3-hydroxyvaleric acid (may be abbreviated as 3HV) in various composition ratios, by changing carbon sources during their culture (Japanese Patent Application Publication (Kokoku) No. 6-15604, Japanese Patent Application Publication (Kokoku) No. 7-14352, Japanese Patent Application Publication (Kokoku) No. 8-19227).
In Japanese Patent Application Laid-Open No. 5-74492, a method in which the copolymer of 3HB and 3HV is produced by bringing Methylobacterium sp., Paracoccus sp., Alcaligenes sp. and Pseudomonas sp. into contact with primary alcohol having three to seven carbons is disclosed.
In Japanese Patent Application Laid-Open No. 5-93049 and Japanese Patent Application Laid-Open No. 7-265065, it is disclosed that binary copolymers of 3HB and 3-hydroxyhexanoic acid (may be abbreviated as 3HHx, hereinafter) are produced by culturing Aeromonas caviae) with oleic acid or olive oil as carbon sources.
In Japanese Patent Application Laid-Open No. 9-191893, it is disclosed that Comamonas acidovoranas IFO 13852 produces polyester having 3HB and 4-hydroxybutyric acid as monomer unit through culture using gluconic acid and 1,4-butandiol as carbon sources.
Also, currently, studies are vigorously carried out as to PHA composed of 3-hydroxyalkanoate (may be abbreviated as 3HA hereinafter) of medium-chain-length: abbreviated as mcl) having up to about twelve carbons. Synthetic pathways of PHA can be classified broadly into two types, and specific examples thereof will be shown in the following (1) and (2).
(1) Synthesis using xcex2 oxidation
In Japanese Patent No. 2642937, it is disclosed that PHA having monomer unit of 3-hydroxyalkanoate having six to twelve carbons is produced by giving acyclic aliphatic hydrocarbons as carbon sources to Pseudomonas oleovorans ATCC 29347. Also, in Appl. Environ. Microbiol, 58 (2), 746 (1992), it is reported that Pseudomonas resinovorans produces polyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxydecanoic acid (amount ratio 1:15:75:9) as monomer unit, with octanoic acid as a sole carbon source, and produces polyester having 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid and 3-hydroxudecanoic acid (amount ratio 8:62:23:7) as monomer unit, with hexanoic acid as a sole carbon source. Here, it is believed that the 3HA monomer unit having chain length larger than that of stock fatty acid are by way of fatty acid synthetic pathway that is described in (2).
(2) Synthesis using fatty acid synthesis routs
In Int. J. Biol. Macromol., 16 (3), 119 (1994), it is reported that Pseudomonas sp.61-3 strain produces polyester having as monomer unit 3-hydroxyalkanoic acids such as 3-hydroxybutyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxydodecanoic acid, and 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-decenoic acid and 3-hydroxy-5-cis-dodecenoic acid, with sodium gluconate as a sole carbon source.
By the way, biosynthesis of PHA is usually performed by PHA synthase using as a matrix xe2x80x9cD-3-hydroxyacyl-CoAxe2x80x9d produced as intermediates of various metabolism pathways in cells.
Here, xe2x80x9cCoAxe2x80x9d refers to xe2x80x9ccoenzyme Axe2x80x9d. And, as described in the above prior art of (1), if using fatty acids such as octanoic acid and nonanoic acid as carbon sources, the biosynthesis of PHA is performed with xe2x80x9cD-3-hydroxyacyl-CoAxe2x80x9d produced in the xe2x80x9cxcex2-oxidation cyclexe2x80x9d as a starting substance.
Reactions through which PHA is biosynthesized by way of the xe2x80x9cxcex2-oxidation cyclexe2x80x9d are shown in the following. 
On the other hand, as described in the above prior art of (2), if PHA is biosynthesized using saccharides such as glucose, the biosynthesis is performed using the xe2x80x9cD-3-hydroxyacyl-CoAxe2x80x9d converted from xe2x80x9cD-3-hydroxyacyl-ACPxe2x80x9d produced in the xe2x80x9cfatty acid synthetic pathwayxe2x80x9d as start substance.
Here, xe2x80x9cACPxe2x80x9d refers to xe2x80x9cacyl carrier proteinxe2x80x9d.
By the way, as described previously, both any PHA being synthesized in above (1) and (2) is PHA composed of monomer unit having alkyl groups on the side chain, that is xe2x80x9cusual PHAxe2x80x9d. However, if considering more widespread application of microorganism producible PHA like this, for example application as functional polymers, it is expected that PHA having substituents other than alkyl groups (e.g. phenyl groups) incorporated in the side chain is extremely useful. Examples of other substituents include unsaturated hydrocarbons, ester groups, allyl groups, cyano groups, halogenated hydrocarbons and epoxide.
For synthesis of PHA having such substituents incorporated therein (hereinafter, referred to as xe2x80x9cunusual PHAxe2x80x9d as necessary), for example, a report as to PHA having aryl groups and the like in terms of synthesis using xcex2 oxidation is found in Macromolecules, 24, p 5256-5260 (1991). Specifically, it is reported that Pseudomonas oleovorans produces 160 mg of PHA per liter of culture medium (the ratio of dry weight to the cell is 31.6%), containing as monomer unit 3HV, 3-hydroxyheptanoic acid, 3-hydroxynonanoic acid, 3-hydroxyundecanoic acid and 3-hydroxy-5-phenylvaleric acid (may be abbreviated as 3HPV, hereinafter) in the amount ratio of 0.6:16.0:41.1:1.7:40.6 using 5-phenylvaleric acid (may be abbreviated as PVA) and nonanoic acid (mole ratio of 2:1 and total concentration of 10 mmol/L), and produces 200 mg of PHA per liter of culture medium (the ratio of dry weight to the cell mass is 39.2%), containing as monomer unit 3HHx, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3HPV in the amount ratio of 7.3:64.5:3.9:24.3 using PVA and octanoic acid (mole ration of 1:1, and total concentration of 10 mmol/L). It is thought that PHA in this report is synthesized principally by way of the xcex2-oxidation from the fact that nonanoic acid and octanoic acid are used.
Related descriptions are also found in Macromol. Chem., 191, 1957-1965 (1990) and Chirality, 3, 492-494 (1991), and the change of polymer properties that is probably caused by contained 3HPV is recognized.
As described above, for microorganism producible PHA, those having various kinds of compositions and structures have been obtained by varying types of microorganisms for use in their production, culture compositions, culture conditions and the like, but they cannot be appropriate yet in terms of properties when their application as plastics is considered. In order to further expand the application range of microorganism producible PHA, it is important to consider the improvement of properties more widely, and for this purpose, research and development of PHA containing monomer unit having more diversified structures, methods for production thereof, and microorganisms capable of producing desired PHA effectively are essential.
On the other hand, for PHA having substituents incorporated in the side chain (unusual PHA) as described above, incorporated substituents are selected in accordance with desired properties and the like, whereby development as xe2x80x9cfunctional polymersxe2x80x9d having very useful functions and properties resulting from properties of incorporated substituents and the like can also be expected, and research and development of excellent PHA allowing such functionality and biodegradability to be compatible with each other, methods for production thereof, and microorganisms capable of producing desired PHA efficiently are also important challenges.
Other examples of PHA having these substituents incorporated in the side chain include PHAs having phenyl groups, and phenoxy groups on the side chain.
As another example of phenyl groups, it is reported that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(4-tril) valeric acid as monomer unit through culture on a medium containing 5-(4-tril) valeric acid (5-(4-methylphenyl) valeric acid), in Macromolecules, 29, 1762-1766 (1996).
Furthermore, in Macromolecules, 32, 2889-2895 (1999), it is reported that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(2,4-dinitrophenyl) valeric acid and 3-hydroxy-5-(4-nitrophenyl) valeric acid as monomer unit through culture on a medium containing 5-(2,4-dinitrophenyl) valeric acid and nonanoic acid.
Also, as an example of phenoxy groups, it is reported that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid as units from 11-phenoxyundecanoic acid, in Macromol. Chem. Phys., 195, 1665-1672 (1994).
Also, in Macromolecules, 29, 3432-3435 (1996), it is reported that using Pseudomonas oleovorans, PHA containing 3-hydroxy-4-phenoxybutyric acid and 3-hydroxy-6-phenoxyhexanoic acid as units is produced from 6-phenoxyhexanoic acid, PHA containing 3-hydroxy-4-phenoxybutyric acid, 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-8-phenoxyoctanoic acid as units is produced from 8-phenoxyoctanoic acid, and PHA containing 3-hydroxy-5-phenoxyvaleric acid and 3-hydroxy-7-phenoxyheptanoic acid as units is produced from 11-phenoxyundecanoic acid. Yields of polymers in this report are extracted and are shown in the following.
Furthermore, in Can. J. Microbiol., 41, 32-43 (1995), PHA containing 3-hydroxy-p-cyanophenoxyhexanoic acid or 3-hydroxy-p-nitrophenoxyhexanoic acid as monomer unit is successfully produced with octanoic acid and p-cyanophenoxyhexanoic acid or p-nitrophenoxyhexanoic acid as a raw material, using Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442.
In Japanese Patent No. 2989175, methods of producing a homopolymer composed of 3-hydroxy-5-(monofluorophenoxy)pentanoate (may be abbreviated as 3H5(MFP)P, hereinafter) units or 3-hydroxy-5-(difluorophenoxy)pentanoate (may be abbreviated as 3H5 (DFP)P, hereinafter) units and a copolymer containing at least 3H5(MFP)P units or 3H5 (DFP) P units; Pseudomonas putida for synthesizing these polymers; and the above described polymers using Pseudomonas sp. are described.
These productions are performed through xe2x80x9ctwo-stage culturexe2x80x9d as described below.
Culture time: first stage, 24 hours ; second stage, 96 hours
Carbon sources and obtained polymers in respective stages are shown below.
(1) Obtained polymer: 3H5 (MFP)P homopolymer
Carbon sources in the first stage: citric acid, yeast extract
Carbon source in the second stage: monofluorophenoxyundecanoic acid
(2) Obtained polymer: 3H5 (DFP)P homopolymer
Carbon sources in the first stage: citric acid, yeast extract
Carbon source in the second stage: difluorophenoxyundecanoic acid
(3) Obtained polymer: 3H5 (MFP) P copolymer
Carbon sources in the first stage: octanoic acid or nonanoic acid, and yeast extract
Carbon source in the second stage: monofluorophenoxyundecanoic acid
(4) Obtained polymer: 3H5 (DFP) P copolymer
Carbon sources in the first stage: octanoic acid or nonanoic acid, and yeast extract
Carbon source in the second stage: difluorophenoxyundecanoic acid
For the effect, fatty acid of medium-chain-length having substituents can be materialized to synthesize a polymer having phenoxy groups with chain ends substituted by one to two fluorine atoms, and stereoregularity and water repellency can be given while maintaining high melting points and good processability.
Also, it is expected that PHA containing cyclohexyl groups in the monomer unit exhibits polymer properties different from those of PHA containing usual aliphatic hydroxyalkanoic acid as units, and an example of its production by Pseudomonas oleovorans has been reported (Macromolecules, 30, 1611-1615 (1997)).
According to this report, when Pseudomonas oleovorans was cultured in a medium where nonanoic acid (may be abbreviated as NA, hereinafter) and crylohexylbutyric acid (may be abbreviated as CHBA, hereinafter) or cyclohexylvaleric acid (may be abbreviated as CHVA, hereinafter) coexisted, PHA containing units containing cyclohexyl groups and units derived from nonanoic acid was obtained (each ratio is unknown).
For its yields and the like, it has been reported that the amount ratio between CHBA and NA was changed with total concentration of 20 mmol/L to obtain results as shown in the following.
In this example, however, the yield of polymers for culture medium is not enough, and obtained PHA itself has also aliphatic hydroxyalkanoic acid derived from nonanoic acid coexisting in its monomer unit.
In this way, in the case where PHA having various substituents incorporated in the side chain is to be produced using microorganisms, methods in which alkanoate having substituents to be incorporated is used not only as polymer material but also as a carbon source for growth are used, as found in the reported examples of Pseudomonas oleovorans described previously.
However, in the method in which alkanoate having substituents to be incorporated is used not only as polymer material but also as a carbon source for growth, the supply of an energy source on the basis of production of acetyl-CoA through xcex2-oxidation from the alkanoate is expected, and in this method, acetyl-CoA cannot be produced through xcex2 oxidation unless starting substance having a certain degree of chain length are used, and alkanoate that can be used as a row material of PHA is thus limited, which is a major problem. Also, since matrixes with chain length reduced by two methylene chains are newly produced through xcex2-oxidation and they are incorporated as monomer unit of PHA, PHA that is synthesized is often a copolymer composed of monomer unit with chain lengths different by two methylene chains. In the reported example described above, a copolymer composed of three kinds of monomer unit of 3-hydroxy-8-phenoxyoctanoic acid derived from 8-phenoxyoctanoic acid, and 3-hydroxy-6-phenoxyhexanoic acid and 3-hydroxy-4-phenoxybutyric acid, which are by-products derived from metabolites is produced. In this respect, in the case of obtaining PHA composed of single monomer unit, it is extremely difficult to use this method. Furthermore, in methods based on the supply of energy sources on the basis of production of acetyl-CoA through xcex2-oxidation, the growth of microorganisms is slow and thus much time is required for synthesis of PHA, and yields of synthesized PHA tends to be lower, which is also a major problem.
Thus, methods in which microorganisms are cultured on a medium where fatty acids of medium-chain-length such as octanoic acid and nonanoic acid and so forth coexist as a carbon source for growth in addition to alkanoate having substituents to be incorporated, and then PHA is extracted are considered effective, and are generally used.
However, according to considerations by the present inventors, PHA synthesized by way of the xcex2-oxidation pathway using fatty acids of medium-chain-length such as octanoic acid and nonanoic acid and so on as a carbon source for growth has lower purity, and 50% or more of obtained polymers are mcl-3HA monomer unit that are monomer unit derived from the carbon source for growth (for example, 3-hydroxyoctanoic acid and 3-hydroxynonanoic acid), namely units of xe2x80x9cusual PHAxe2x80x9d. These mcl-3HA units are sticky polymers at room temperature in the case of single composition, and if they coexist in large quantity in PHA intended by the present invention, the glass transition temperature (Tg) of the polymer is significantly decreased. Thus, when hard polymer properties are to be obtained at room temperature, the coexistence of the mcl-3HA monomer unit is not desirable. Also, a hetero side chain structure like this is known to hinder interaction derived from the intramolecular or intermolecular side chain structure and have a significant influence on crystalline and orientation. For achieving improvement of polymer properties and addition of new functions, the coexistence of these mcl-3HA monomer unit is a major problem. As means for solving this problem, a purification process for separating and removing xe2x80x9cundesiredxe2x80x9d monomer unit such as mcl-3HA monomer unit derived from the carbon source for growth is provided to obtain PHA composed of only monomer unit having specific substituents. However, the problem is that operations become complicated and significant reduction of yields cannot be avoided. The more serious problem is that it is extremely difficult to remove only undesired monomer unit when desired monomer unit and undesired monomer unit form a copolymer. In particular, when the object is to synthesize PHA containing monomer unit having groups obtained from unsaturated hydrocarbons, ester groups, allyl groups, cyano groups, nitro groups, groups obtained from halogenated hydrocarbons, and groups with epoxide, etc. incorporated therein as a side chain, there are many cases where the mcl-3HA monomer unit forms a copolymer with the desired monomer unit, and the removal of the mcl-3HA monomer unit after synthesis of PHA is thus extremely difficult.
Thus, the present inventors have reached recognition that development of a biosynthetic method by which xe2x80x9cunusual PHAxe2x80x9d can be obtained in high purity is absolutely necessary when considering application to functional polymers. Therefore, it has been thought that development of excellent polymers having both functionality and biodegradability and microorganisms capable of producing such polymers and accumulating them in the cell as described above, and a method for efficiently biosynthesizing such PHA in high purity is quite useful and important.
The present invention solves the above described problems, and is to provide PHA containing monomer unit of diversified structures having in the side chain substituents useful as device material and medical material (unusual PHA) and provide a method of producing such xe2x80x9cunusual PHAxe2x80x9d using microorganisms, and in particular, provide a producing method in which coexisting undesired monomer unit are reduced and desired xe2x80x9cunusual PHAxe2x80x9d can be thus obtained in high purity, and in addition, high fields are achieved.
Thus, the present inventors have continued to carry out enthusiastic studies on screening of microorganisms capable of producing various kinds of PHA and accumulating them in the cell and a method of producing desired PHA using these microorganisms, with the aim of developing PHA having in the side chain functional groups useful as device material and medical material. As a result, we have found microorganisms capable of producing novel PHA containing as monomer unit 3-hydroxythienylalcanoic acid represented by Chemical Formula [2], 
(n is any one of integers of 1 to 8) using as a stock thienylalkanoic acid represented by Chemical Formula [9], 
(n is any one of integers of 1 to 8) and accumulating the same in the cell, and in addition, we have found that such PHA can be biosynthesized by culturing these microorganisms under coexistence of thienylalkanoic acid represented by the above described Chemical Formula [9] and saccharides, yeast extract or polypeptone, and that the PHA obtained thereby has higher purity.
More particularly, we have found microorganisms capable of producing novel PHA containing as monomer unit 3-hydroxy-4-(2-thienyl) butyric acid (hereinafter abbreviated as 3HTB) represented by Chemical Formula [5], 
3-hydroxy-5-(2-thienyl) valeric acid (hereinafter abbreviated as 3HTV) represented by Chemical Formula [6], 
3-hydroxy-6-(2-thienyl) hexanoic acid (hereinafter abbreviated as 3HTHx) represented by Chemical Formula [7], 
and 3-hydroxy-7-(2-thienyl) heptanoic acid (hereinafter abbreviated as 3HTHp) represented by Chemical Formula [8], 
using as stocks at least any one of 5-(2-thienyl) valeric acid (hereinafter abbreviated as TVA) represented by Chemical Formula [11], 
6-(2-thienyl) hexanoic acid (hereinafter abbreviated as THxA) represented by Chemical Formula [12], 
7-(2-thienyl) heptanoic acid (hereinafter abbreviated as THpA) represented by Chemical Formula [13], 
and accumulating the same in the cell, and in addition we have found that such PHA can be biosynthesized by culturing these microorganisms under coexistence of TVA, THxA or THpA and saccharides, yeast extract or polypeptone, and that the PHA obtained thereby has higher purity, resulting in the present invention.
That is, the present invention relates to polyhydroxyalkanoate having monomer unit represented by Chemical Formula [2]
(n is any one of integers of 1 to 8).
Also, the present invention relates to a method of producing the PHA, characterized by having a process in which microorganisms are cultured on a culture medium containing thienylalkanoic acid represented by Chemical formula [9], 
(n is any one of integers of 1 to 8), thereby making such microorganisms produce PHA having corresponding monomer unit represented by the above described Chemical Formula [10], 
(wherein in the above formula, m is one or more selected from the group consisting of n, nxe2x88x922, nxe2x88x924 and nxe2x88x926, and also an integer greater than or equal to 1).
That is, the method of producing PHA according to the present invention is characterized by having a process of culturing microorganisms producing PHA containing 3HTB, 3HTV, 3HTHx or 3HTHp as monomer unit under the coexistence of TVA, THxA or THpA and saccharides, yeast extract or polypeptone.
According to the present invention, new polyhydroalkanoate having monomer unit represented by the above described Chemical Formula [1], and a method of producing the polyhydroalkanoate using microorganisms are provided. By this, the polyhydroalkanoate useful as a functional polymer can be efficiently produced, and its application to various fields such as device material and medical material can be expected.
According to the present invention, it is made possible to provide PHA (unusual PHA) containing monomer unit of diversified structures having on the side chain substituents useful as device material, medical material and the like, and provide a method of producing the xe2x80x9cunusual PHAxe2x80x9d using microorganisms. Particularly, a method in which undesired monomer unit are reduced and thus desired xe2x80x9cunusual PHAxe2x80x9d can be obtained in high purity, and high yields are achieved can be provided.